The overall objective of this research proposal is to define at the[unreadable] molecular level the individual steps in reverse transcription of the Human immunodeficiency virus (HIV-l)[unreadable] viral genome. The experimental approach proposed here is unique because it[unreadable] uses viruses derived from mutant proviral genomes to study reverse[unreadable] transcription. Three key areas of reverse transcription, for which there[unreadable] is very little information, will be investigated. First, the isolated[unreadable] virions from HIV-l infected cells contain the necessary viral proteins for[unreadable] reverse transcription. How the virus assembles these components into the[unreadable] virion remains a major question. Second, the initiation of reverse[unreadable] transcription requires a cellular tRNA-Lys as aprimer and occurs at a[unreadable] region of the viral RNA genome, the primer binding site (PBS). How the[unreadable] viral proteins recognize the PBS and how the tRNA-Lys is selected for[unreadable] inclusion into the virion is unknown. Third, the process of reverse[unreadable] transcription requires template switching by the reverse transcriptase.[unreadable] How this occurs at the molecular level is not understood. To address these[unreadable] questions, the following specific aims are proposed:[unreadable] 1. To define the roles of the Gag-Pol polyprotein in reverse[unreadable] transcription. The reverse transcriptase is incorporated into the virion[unreadable] in the form of a larger precursor molecule, pr160(gag-Pol). The regions of[unreadable] Gag-Pol which are required for incorporation into virions, and the[unreadable] interaction between Gag-Pol and the cellular tRNA-Lys will be analyzed[unreadable] using a complementation system developed by this laboratory. We have[unreadable] demonstrated that the unprocessed Gag-Pol polyprotein contains reverse[unreadable] transcriptase activity and the function of this activity in initiation of[unreadable] reverse transcription will be investigated.[unreadable] 2. To determine the structural features of the viral RNA genome containing[unreadable] the PBS required for initiation of reverse transcription. The minimum[unreadable] number of nucleotides within the PBS and the region of the viral RNA[unreadable] genome encompassing the PBS which is required for initiation of reverse[unreadable] transcription will be determined.[unreadable] 3. To characterize features of the viral genome required for template[unreadable] switching in reverse transcription. Mutations in the PBS will be[unreadable] constructed to investigate the requirements for template switching. In[unreadable] addition, the role of reverse transcriptase in promoting template[unreadable] switching will be analyzed.[unreadable] Reverse transcription is an early step in viral replication and is a major[unreadable] target for anti-viral drugs. The long-term goal of this research is to[unreadable] delineate the in vivo details of reverse transcription which will provide[unreadable] new targets for therapeutic strategies to inhibit this critical step in[unreadable] HIV-l replication.[unreadable]